Back to article: Cellular cholesterol licenses Legionella pneumophila intracellular replication in macrophages

FIGURE 5: Effects of U18666A and ketoconazole on Lp uptake, dissemination and intracellular replication. (a,c-f) Live-cell microscopy analysis of BMDMs infected with GFP+ Lp ΔflaA for 62 hr. (a) Representative images (0.85 X 0.63mm, 0.54mm2) acquired at the indicated time points showing individual LCVs containing GFP-expressing Lp in a monolayer of infected BMDMs (∼1,000 cells/image) which were treated with either DMSO, 3.3 µM ketoconazole or 5 µM U18666A. (b) Micrographs showing representative LCVs that support bacterial replication in BMDMs infected with GFP+ Lp ΔflaA at twelve hpi and treated as indicated. (c) Distribution of LCV sizes (µm2) harbored by BMDMs at eight and 52 hpi. Cells were treated as indicated. The size of each bin is 4µm2. Cumulative data from twelve images acquired from three technical replicates for each treatment and each time-point are graphed as LCV counts/bin. (d) The average number of LCVs per image, (e) the average LCV size (µm2), and (f) the number of dead BMDMs are shown over the course of the infection. BMDMs were treated as indicated. Averages ± SD of data from twelve images from three technical replicates (four images per replicate) for each condition are shown. For statistical analysis, each data series was compared to the data series obtained from DMSO-treated cells using two-way ANOVA analysis. **** p<0.0001; *** p<0.0002; ** p<0.0021; * p< 0.033 (f) In control treatments, BMDMs were treated either with 0.2% Triton X-100 to determine the maximum number of dead cells or with the cytotoxic reagent gemcitabine. (a-f) Representative data from one of three biological replicates are shown.

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